Nano-bioprotectant dsRsGH1@ε-PL@CMCS enhances RNAi efficacy against Rhizoctonia solani via dual mechanisms: Interacting with the vesicle transport protein SEC22 and modulating the tobacco phyllosphere microbiota

Nanocarrier-mediated delivery of double-stranded RNA (dsRNA) is a promising strategy for plant disease control. Our previous study has shown that the self-assembled ε-poly- l -lysine/carboxymethyl chitosan (ε-PL@CMCS) nanocarrier significantly improves the efficiency of RNA interference (RNAi) against Rhizoctonia solani AG3 TB. However, the molecular mechanisms underlying this enhancement and the potential ecological impacts on phyllosphere microbial communities remain unclear. In this study, we confirmed that ds RsGH1 @ε-PL@CMCS had no adverse effects on the growth and development of plants. Transcriptome analysis revealed that DEGs were significantly enriched in ‘SNARE interactions in vesicular transport’ pathway. Among them, ds RsGH1 @ε-PL@CMCS specifically upregulate the vesicle transport protein SEC22 in Nicotiana tabacum. Surface plasmon resonance (SPR) assay demonstrated that the ds RsGH1 @ε-PL@CMCS binds to SEC22 protein with 7.6-fold higher affinity than that of the naked ds RsGH1 . Silencing SEC22 in the ds RsGH1 @ε-PL@CMCS treatment reduced the silencing efficiency of RsGH1 from 53.3% to 39%, confirming its essential role. Concurrently, high-throughput sequencing revealed that SIGS treatments did not disrupt bacterial community structure but increased fungal diversity and suppressed the pathogen, while enriching beneficial fungi such as Tulasnella . Our findings elucidate a molecular link between nano-bioprotectant ds RsGH1 @ε-PL@CMCS-enhanced RNAi and host vesicle transport, while confirming the biocompatible and microbiome-modulating potential. This provides a dual perspective for developing efficient and sustainable RNAi-based strategies for protecting plants.